scholarly journals Inositol phospholipid arachidonic acid metabolism in GH3 pituitary cells

1986 ◽  
Vol 236 (1) ◽  
pp. 235-242 ◽  
Author(s):  
D T Dudley ◽  
A A Spector
Keyword(s):  
Arachidonic Acid ◽  
Molecular Species ◽  
Arachidonic Acid Metabolism ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Trh Stimulation ◽  
Inositol Phospholipids ◽  
Rat Pituitary ◽  
Arachidonic Acids ◽  
Phosphatidylinositol 4

Inositol phospholipids in cultured GH3 cells, a prolactin secreting, thyrotropin-releasing hormone (TRH) sensitive rat pituitary cell line, exhibit a preferential selectivity for incorporating arachidonic acid. Fatty acid composition data show that all inositol phospholipids are enriched in stearic and arachidonic acids to a much greater degree than other cellular phospholipids. Incubation of GH3 cells with radioactive stearate, oleate, arachidonate, eicosapentaenoate or docosahexaenoate also showed that much more stearate and arachidonate were incorporated into inositol phospholipids. In short term incubations with tracer amounts of radioactive arachidonate, incorporation was initially into phosphatidylinositol (PtdIns), with phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] being labelled at later times. During longer incubations, all of the inositol phospholipids reach equilibrium at about 10 h, and the resulting specific activities of the three fractions were similar. These findings suggest that arachidonate is incorporated initially into PtdIns and that PtdIns is then phosphorylated. There was no release of either arachidonate or eicosanoid products when GH3 cells were incubated with TRH. However, TRH stimulation of 32P-labelled GH3 cells resulted in rapid breakdown of PtdIns(4,5)P2 and PtdIns4P, with concomitant increases in [32P]phosphatidic acid and [32P]PtdIns. When the [32P]PtdIns was further analysed by argentation chromatography to separate PtdIns molecular species, it was found that tetraenoic (stearate/arachidonate) species accounted for 80% of the stimulated labelling. The selectivity for arachidonate incorporation into inositol phospholipids coupled with turnover of the arachidonate-containing molecular species suggests that inositol phospholipids containing arachidonic acid or the diacylglycerol resulting therefrom may play a vital cellular role in GH3 cells. This role may involve the operation of the PtdIns cycle itself rather than a stimulated release of arachidonate for eicosanoid formation.

Arachidonic acid mobilizes calcium and stimulates prolactin secretion from GH3 cells

1984 ◽  
Vol 246 (5) ◽  
pp. E458-E462 ◽  
Author(s):  
R. N. Kolesnick ◽  
I. Musacchio ◽  
C. Thaw ◽  
M. C. Gershengorn
Keyword(s):  
Arachidonic Acid ◽  
Saturated Fatty Acids ◽  
Arachidonic Acid Metabolism ◽  
Prolactin Secretion ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Alpha Linolenic Acid ◽  
Rat Pituitary ◽  
Experimental Approaches ◽  
Rat Pituitary Cells

Because arachidonic acid and/or its metabolites may be intracellular effectors of calcium-mediated secretion, we studied whether arachidonic acid added exogenously mobilizes calcium and stimulates prolactin secretion from GH3 cells, cloned rat pituitary cells. Arachidonic acid caused efflux of 45Ca from preloaded cells and stimulated prolactin secretion. The concentration dependencies of these effects were similar; stimulation was attained with 3 microM arachidonic acid. To determine indirectly whether these effects may be caused by arachidonic acid itself, not via conversion to metabolites, two experimental approaches were used. First, inhibitors of arachidonic acid metabolism, eicosatetraynoic acid and indomethacin, did not inhibit arachidonic acid-induced prolactin secretion. And second, alpha-linolenic acid, which cannot be converted to arachidonic acid, and linoleic acid, but not saturated fatty acids of equal chain length, stimulated 45Ca efflux and prolactin secretion. These data demonstrate that arachidonic acid added exogenously causes Ca2+ mobilization and prolactin secretion from GH3 cells and suggest that arachidonic acid itself, not via metabolism, may be a cellular regulator of prolactin secretion.

scholarly journals Depletion of arachidonic acid from GH3 cells. Effects on inositol phospholipid turnover and cellular activation

1987 ◽  
Vol 246 (3) ◽  
pp. 669-679 ◽  
Author(s):  
D T Dudley ◽  
D E Macfarlane ◽  
A A Spector
Keyword(s):  
Arachidonic Acid ◽  
Cell Activation ◽  
Gh3 Cells ◽  
Arachidonic Acid Content ◽  
Cellular Activation ◽  
Inositol Phospholipids ◽  
Rat Pituitary ◽  
Long Time ◽  
Transient Events

We have adapted rat pituitary GH3 cells to grow in delipidated culture medium. In response, esterfied linoleic acid and arachidonic acid become essentially undetectable, whereas eicosa-5,8,11-trienoic acid accumulates and oleic acid increases markedly. These changes occur in all phospholipid classes, but are particularly pronounced in inositol phospholipids, where the usual stearate/arachidonate profile is replaced with oleate/eicosatrienoate (n − 9) and stearate/eicosatrienoate (n − 9). Incubation of arachidonate-depleted cells with 10 microM-arachidonic acid for only 24 h results in extensive remodelling of phospholipid fatty acids, such that close-to-normal compositions and arachidonic acid content are achieved for the inositol phospholipids. In comparison studies with arachidonic acid-depleted or -repleted cells, it was found that the arachidonate content does not affect thyrotropin-releasing-hormone (TRH)-stimulated responses measured at long time points, including [32P]Pi labelling of phosphatidylinositol and phosphatidic acid, stimulation of protein phosphorylation, and basal or TRH-stimulated prolactin release. However, transient events such as stimulated breakdown of inositol phospholipids and an initial rise in diacylglycerol are enhanced by the presence of arachidonate. These results show that arachidonic acid itself is not required for operation of the phosphatidylinositol cycle and is not an obligatory intermediate in TRH-mediated GH3 cell activation. It is possible that any structural or functional role of arachidonic acid in these processes is largely met by replacement with eicosatrienoate (n − 9). However, since arachidonate in inositol phospholipids facilitates their hydrolysis upon stimulation by TRH, arachidonic acid apparently may have a specific role in the recognition of these lipids by phospholipase C.

1,25-Dihydroxyvitamin D3-induced upregulation of the thyrotropin-releasing hormone receptor in clonal rat pituitary GH3 cells

1995 ◽  
Vol 147 (3) ◽  
pp. 397-404 ◽  
Author(s):  
L M Atley ◽  
N Lefroy ◽  
J D Wark
Keyword(s):  
Vitamin D ◽  
Fold Increase ◽  
Thyrotropin Releasing Hormone ◽  
Gh3 Cells ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Hormone Production ◽  
Releasing Hormone ◽  
Rat Pituitary ◽  
Trh Receptor

Abstract 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is active in primary dispersed and clonal pituitary cells where it stimulates pituitary hormone production and agonist-induced hormone release. We have studied the effect of 1,25-(OH)2D3 on thyrotropin-releasing hormone (TRH) binding in clonal rat pituitary tumour (GH3) cells. Compared with vehicle-treated cells, 1,25-(OH)2D3 (10 nmol/l) increased specific [3H]MeTRH binding by 26% at 8 h, 38% at 16 h, 35% at 24 h and reached a maximum at 48 h (90%). In dose–response experiments, specific [3H]MeTRH binding increased with 1,25-(OH)2D3 concentration and reached a maximum at 10 nmol/l. Half-maximal binding occurred at 0·5 nmol 1,25-(OH)2D3/l. The vitamin D metabolite, 25-OH D3, increased [3H]MeTRH binding but was 1000-fold less potent than 1,25-(OH)2D3. In equilibrium binding assays, treatment with 10 nmol 1,25-(OH)2D3/l for 48 h increased the maximum binding from 67·4 ± 8·8 fmol/mg protein in vehicle-treated cells to 96·7 ± 12·4 fmol/mg protein in treated cells. There was no difference in apparent Kd (1·08 ± 0·10 nmol/l for 1,25-(OH)2D3-treated and 0·97 ± 0·11 nmol/l for vehicle-treated cells). Molecular investigations revealed that 10 nmol 1,25-(OH)2D3/l for 24 h caused an 8-fold increase in TRH receptor-specific mRNA. Actinomycin D (2 μg/ml, 6 h) abrogated the 1,25-(OH)2D3-induced increase in [3H]MeTRH binding. Cortisol also increased [3H]MeTRH binding but showed no additivity or synergism with 1,25-(OH)2D3. TRH-stimulated prolactin release was not enhanced by 1,25-(OH)2D3. We conclude that the active vitamin D metabolite, 1,25-(OH)2D3, caused a time- and dose-dependent increase in [3H]MeTRH binding. The effect was vitamin D metabolite-specific and resulted from an upregulation of the TRH receptor. Further studies are needed to determine the functional significance of this novel finding. Journal of Endocrinology (1995) 147, 397–404

scholarly journals Detection of two chromatin proteins which bind specifically to the 5'-flanking region of the rat prolactin gene.

1985 ◽  
Vol 5 (11) ◽  
pp. 2967-2974 ◽  
Author(s):  
B A White ◽  
G M Preston ◽  
T C Lufkin ◽  
C Bancroft
Keyword(s):  
Transcription Initiation ◽  
Genomic Clone ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Sequence Specificity ◽  
Strong Binding ◽  
Prolactin Gene ◽  
Rat Pituitary ◽  
Chromatin Proteins ◽  
Flanking Region

We employed a protein gel blotting procedure to search for nuclear proteins from rat pituitary cells that bind preferentially to the 5'-flanking region of the rat prolactin gene. By gel blots of chromatin proteins from GH3 rat pituitary tumor cells with a 32P-labeled prolactin genomic clone, we detected two major binding proteins with molecular weights of approximately 44,000 and 48,000, designated NP44 and NP48, respectively. Both NP44 and NP48 are minor chromatin proteins which are extracted at low salt concentrations (0.4 M NaCl) and exhibit a range of slightly acidic isoelectric variants. NP44 and NP48 were detected at similar levels in chromatin extracts of GH3 cells, the prolactin-negative GC cell variant of the GH3 cells, and normal rat pituitary tissue. Considerably lower levels of these two proteins were found in chromatin extracts from rat liver and rat C6 glial cells. NP44 and NP48 exhibit DNA sequence specificity, as evidenced by their strong binding to the upstream flanking region of the prolactin gene, but only very weak binding to plasmid DNA, rat prolactin or growth hormone cDNAs, or upstream flanking regions of two other rat genes. By analyzing subclones of a rat prolactin genomic clone, we established that NP44 and NP48 bind to at least two sites, which are located between 0.4 and 2.0 kilobases (region I) and between 2.0 and 4.8 kilobases (region II) upstream of the transcription initiation site. These findings are discussed in the context of a possible functional association between the strong binding of NP44 and NP48 to the prolactin 5'-flanking region and pituitary-specific expression of the prolactin gene.

172 IN VITRO EXPOSURE TO XENOESTROGENS INDUCES GROWTH HORMONE TRANSCRIPTION AND RELEASE VIA AN ESTROGEN RECEPTOR-DEPENDENT PATHWAY IN RAT PITUITARY GH3 CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 166
Author(s):  
V.-H. Dang ◽  
E.-B. Jeung
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Estrogen Receptor ◽  
Gene Transcription ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Rat Pituitary ◽  
In Vitro Exposure ◽  
Gh Gene

The term endocrine disruptor (ED) has been used widely to characterize natural and synthetic environmental compounds that may interfere with the endocrine system(s) of humans and wildlife. In previous studies, we demonstrated that in vitro single exposure to EDs induces CaBP-9k expression, a useful biomarker for detecting the estrogenic activities of EDs in rat pituitary GH3 cells. Here we employ the identical model to examine the effects of EDs in the regulation of growth hormone (GH) gene expression, an important hormone in growth, development, and body composition. We measured levels of GH mRNA transcription and GH release using semi-quantitative RT-PCR and EIA kit, respectively. GH3 cells were treated with alkyphenols (APs), i.e., octyl-phenol (OP), nonyl-phenol (NP), and bisphenol A (BPA), in a dose-dependent manner (10–5, 10–6, and 10–7 M) and harvested following 24 h of treatment. Cells were also exposed to a high concentration (10–5 M) of OP, NP, or BPA and harvested at various time points (1, 3, 6, 12, and 24 h). An anti-estrogen, ICI 182780 (10–7 M) was used to examine the potential involvement of estrogen receptor (ER) in the induction of GH by EDs through an ER-mediated pathway. The data were analyzed by one-way ANOVA, followed by Tukey's multiple comparison. OP, NP, and BPA induced a significant increase in GH gene expression at high (10–5 M) and medium (10–6 M) doses at 24 h. ED-exposure induced a marked increase in GH gene transcription as early as 6 h and peaked at 12 h. Co-treatment with ICI 182780 significantly attenuated ED-induced GH expression in GH3 cells. Interestingly, the level of in vitro GH release was increased significantly at 24 h in response to OP, NP, or BPA, whereas co-treatment with ICI 182780 significantly diminished ED-induced GH secretion in GH3 cells, indicating that ER may play a part in both GH gene transcription and GH release in these cells. Here we demonstrate for the first time that single in vitro exposure to OP, NP, or BPA results in an increase in GH expression at 24 h in GH3 rat pituitary cells. These results may provide new insight into the mode of ED action in GH gene regulation as well as the biological pathway underlying these molecular events. Furthermore, data showing GH responsiveness evoked by EDs supports the aim to develop an assay for use in predicting adverse health effects of EDs in humans and wildlife.

Characterization of a hyperpolarization-activated cation current in rat pituitary cells

1997 ◽  
Vol 272 (3) ◽  
pp. E405-E414 ◽  
Author(s):  
S. M. Simasko ◽  
S. Sankaranarayanan
Keyword(s):  
Primary Cultures ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Sodium Permeability ◽  
Cation Current ◽  
Whole Cell Patch Clamp ◽  
Bath Application ◽  
Rat Pituitary ◽  
Rat Pituitary Cells

Whole cell patch-clamp techniques were used on clonal pituitary cells (GH3) and primary cultures of somatotrophs and lactotrophs to study currents that would be active at or below voltages for the threshold for action potential generation. When GH3 cells were held at -60 mV and pulsed to -120 mV, a slow-activating sustained inward current was observed (-16.5 +/- 1.5 pA in physiological baths, n = 72; approximately 1 s to half-maximal activation, voltage for 50% activation - 101 mV). The current was insensitive to bath application of 10 mM tetraethylammonium, 10 mM 4-aminopyridine, and 1 mM barium but was completely blocked by 3 mM cesium. The current was found to be a mixed cation current with a sodium permeability of 0.29 relative to potassium. These properties indicate that the current belongs to the hyperpolarization-activated cation current (Ih), or I(f), family of currents. However, the current was not altered by the addition of adenosine 3',5'-cyclic monophosphate (cAMP) to the pipette or forskolin to the bath. A similar but smaller current was observed in 15 of 16 somatotrophs but in only 1 of 9 lactotrophs. Application of cesium to spontaneously spiking GH3 cells or somatotrophs had no effect. However, cesium did block an inward holding current observed at -80 mV. These results demonstrate that the I(h) in pituitary cells does not serve as a pacemaking current but suggest that it may influence membrane potential responses when somatotrophs become hyperpolarized.

Detection of two chromatin proteins which bind specifically to the 5'-flanking region of the rat prolactin gene

1985 ◽  
Vol 5 (11) ◽  
pp. 2967-2974
Author(s):  
B A White ◽  
G M Preston ◽  
T C Lufkin ◽  
C Bancroft
Keyword(s):  
Transcription Initiation ◽  
Genomic Clone ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Sequence Specificity ◽  
Strong Binding ◽  
Prolactin Gene ◽  
Rat Pituitary ◽  
Chromatin Proteins ◽  
Flanking Region

We employed a protein gel blotting procedure to search for nuclear proteins from rat pituitary cells that bind preferentially to the 5'-flanking region of the rat prolactin gene. By gel blots of chromatin proteins from GH3 rat pituitary tumor cells with a 32P-labeled prolactin genomic clone, we detected two major binding proteins with molecular weights of approximately 44,000 and 48,000, designated NP44 and NP48, respectively. Both NP44 and NP48 are minor chromatin proteins which are extracted at low salt concentrations (0.4 M NaCl) and exhibit a range of slightly acidic isoelectric variants. NP44 and NP48 were detected at similar levels in chromatin extracts of GH3 cells, the prolactin-negative GC cell variant of the GH3 cells, and normal rat pituitary tissue. Considerably lower levels of these two proteins were found in chromatin extracts from rat liver and rat C6 glial cells. NP44 and NP48 exhibit DNA sequence specificity, as evidenced by their strong binding to the upstream flanking region of the prolactin gene, but only very weak binding to plasmid DNA, rat prolactin or growth hormone cDNAs, or upstream flanking regions of two other rat genes. By analyzing subclones of a rat prolactin genomic clone, we established that NP44 and NP48 bind to at least two sites, which are located between 0.4 and 2.0 kilobases (region I) and between 2.0 and 4.8 kilobases (region II) upstream of the transcription initiation site. These findings are discussed in the context of a possible functional association between the strong binding of NP44 and NP48 to the prolactin 5'-flanking region and pituitary-specific expression of the prolactin gene.

The effects of hypothalamic extracts and TRH on the growth and hormone production of GH3 cells in culture

1983 ◽  
Vol 104 (3) ◽  
pp. 287-294 ◽  
Author(s):  
Yukiko Yajima ◽  
Toshikazu Saito
Keyword(s):  
Dna Synthesis ◽  
Gel Filtration ◽  
Control Culture ◽  
Gh3 Cells ◽  
Thymidine Uptake ◽  
Pituitary Cells ◽  
Hormone Production ◽  
Hormone Synthesis ◽  
Rat Pituitary ◽  
Rat Pituitary Cells

Abstract. Hypothalamic factors were tested for their effects on the production of hormones and the growth of GH3 cells, cloned rat pituitary cells producing prolactin (Prl) and growth hormone (GH). Hypothalamic extracts (HE) (0.05 mg/ml) and TRH (0.3 μm) stimulated the synthesis of Prl to levels of 306% and 360%, respectively, of the control culture in a medium containing 0.5% foetal bovine serum (FBS) during a 24 h incubation. They did not affect the rate of GH production. The thymidine uptake was suppressed to 57% and 46% of the control by the addition of HE and TRH, respectively. They also inhibited the growth of GH3 to 70% and 74% of the control culture during an 8-day incubation period. On the other hand, LRH affected neither the rate of hormone production nor the thymidine uptake. Somatostatin suppressed the synthesis of Prl and GH, but it did not affect the incorporation of thymidine into the cells. The gel filtration studies of HE revealed that the inhibitory effects of HE on the thymidine uptake were dependent on two substances, TRH and an unknown factor(s) of high molecular nature. The relationship between hormone synthesis and DNA synthesis will be discussed on the basis of the TRH-induced effects on Prl production and DNA synthesis in GH3 cells.

scholarly journals Differential regulation by phosphatidylinositol 4,5-bisphosphate of pituitary plasma-membrane and cytosolic phosphoinositide kinases

1986 ◽  
Vol 240 (2) ◽  
pp. 341-348 ◽  
Author(s):  
A Imai ◽  
M J Rebecchi ◽  
M C Gershengorn
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Product Inhibition ◽  
Differential Regulation ◽  
Gh3 Cells ◽  
Maximal Effect ◽  
Phosphatidylinositol Kinase ◽  
Rat Pituitary ◽  
Phosphoinositide Kinase ◽  
Phosphatidylinositol 4

Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.

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